3D BIOPRINTED HUMAN ENDOTHELIAZED ADIPOSE TISSUE AS A NEW PREDICTIVE MODEL FOR IN VITRO EVALUATION
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Presented by: AMELIE THEPOT
Background
Human skin is a complex organ harbouring multiple layers, different systems and several distinct cell populations. Current available tissue-engineered skin models often only incorporate the epidermal and dermal layers, and lack of the hypodermis contributing to the mechanical and endocrine properties of the normal human skin. The hypodermis homeostasis is maintained thanks to a pool of precursor cells precommitted to differentiate into adipocytes: the adipose stem cells (ASCs). These cells are essential because mature adipocytes do not proliferate.
Although conventional tissue-engineering strategies led to the development in the past few decades of first generation of engineered hypodermis, such processes are usually time consuming, limited in the shelf-life and confined to the production of constructs having mostly flat and pre-determined geometry. To overcome these limitation of conventional hypodermis tissue-engineering schemes, 3D printing combined with advanced tissue engineering represent a promising approach to reconstruct layer by layer a functional 3D human adipose tissue.
Using our previous findings, the aim this work was to perfect an in vitro 3D bioprinted model of human adipose tissue including a pre-vascularisation by co-bioprinting endothelial cells and ASC.
Experimental Results
To conduct such research, adipose-derived stem cells combined with primary human hypodermal microvascular endothelial cells were mixed in a patented bioink composed of fibrinogen, alginate and gelatin and printed with optimal printing conditions and printer functions. To allow the development of the microvascularisation of the bioprinted adipose tissue, the bioink was functionalized with a cocktail of crucial growth factors to maintain endothelial cells functionality. When applying the favorable printing technique and conditions, viable tissues were obtained, demonstrating high adipogenic differentiation and capillary-like network formation after 21 days of culture. Histological analysis showed that the printed adipose tissue was morphogically consistent with mature adipocyte cells forming droplets. More importantly, immunohistological analysis revealed the formation of an endothelial microvascular network expressing CD31 homogeneously distributed within the bioprinted adipose tissue. Confocal microscopy analysis further showed the formation of capillary-like structures with a clear lumen. Interestingly, the functionalization of the bioink with growth factors demonstrated similar results compared with their supplementation directly in the cell culture medium. Adipogenic modulators such as caffeine and oleic acid promoted lipolysis and lipogenesis, respectively, of adipocytes by modulating lipid content and adipocyte size, demonstrating the functionality of the 3D bioprinted model in vitro.
Conclusion
For the first time, we have developed a bioprinting method for the generation of endotheliazed human adipose tissue with cellular and molecular characteristics closely resembling native human tissue. This unique in vitro model may be a relevant tool to explore molecular mechanisms underlying adipogenesis and to identify dermo-cosmetic active compounds.
Human skin is a complex organ harbouring multiple layers, different systems and several distinct cell populations. Current available tissue-engineered skin models often only incorporate the epidermal and dermal layers, and lack of the hypodermis contributing to the mechanical and endocrine properties of the normal human skin. The hypodermis homeostasis is maintained thanks to a pool of precursor cells precommitted to differentiate into adipocytes: the adipose stem cells (ASCs). These cells are essential because mature adipocytes do not proliferate.
Although conventional tissue-engineering strategies led to the development in the past few decades of first generation of engineered hypodermis, such processes are usually time consuming, limited in the shelf-life and confined to the production of constructs having mostly flat and pre-determined geometry. To overcome these limitation of conventional hypodermis tissue-engineering schemes, 3D printing combined with advanced tissue engineering represent a promising approach to reconstruct layer by layer a functional 3D human adipose tissue.
Using our previous findings, the aim this work was to perfect an in vitro 3D bioprinted model of human adipose tissue including a pre-vascularisation by co-bioprinting endothelial cells and ASC.
Experimental Results
To conduct such research, adipose-derived stem cells combined with primary human hypodermal microvascular endothelial cells were mixed in a patented bioink composed of fibrinogen, alginate and gelatin and printed with optimal printing conditions and printer functions. To allow the development of the microvascularisation of the bioprinted adipose tissue, the bioink was functionalized with a cocktail of crucial growth factors to maintain endothelial cells functionality. When applying the favorable printing technique and conditions, viable tissues were obtained, demonstrating high adipogenic differentiation and capillary-like network formation after 21 days of culture. Histological analysis showed that the printed adipose tissue was morphogically consistent with mature adipocyte cells forming droplets. More importantly, immunohistological analysis revealed the formation of an endothelial microvascular network expressing CD31 homogeneously distributed within the bioprinted adipose tissue. Confocal microscopy analysis further showed the formation of capillary-like structures with a clear lumen. Interestingly, the functionalization of the bioink with growth factors demonstrated similar results compared with their supplementation directly in the cell culture medium. Adipogenic modulators such as caffeine and oleic acid promoted lipolysis and lipogenesis, respectively, of adipocytes by modulating lipid content and adipocyte size, demonstrating the functionality of the 3D bioprinted model in vitro.
Conclusion
For the first time, we have developed a bioprinting method for the generation of endotheliazed human adipose tissue with cellular and molecular characteristics closely resembling native human tissue. This unique in vitro model may be a relevant tool to explore molecular mechanisms underlying adipogenesis and to identify dermo-cosmetic active compounds.